This project proposes to study by means of optical techniques distinct physiological steps in the normal excitation - contraction (E-C) coupling sequence of striated muscle cells. Optical signals have been selected for study because of a presumed specificity for a particular step in E-C coupling. In general, such steps are not accessible in the intact fiber to study by more conventional electrophysiological techniques. For example, (i) potential changes across surface, transverse tubular, or sarcoplasmic reticulum (SR) membranes may be monitored by changes in intrinsic birefringence or extrinsic fluorescence and absorbance; (ii) Ca++ entry into the myoplasm may be monitored by absorbance changes of indicator dyes such as Arsenazo III and Antipyrylazo III that have been injected into the myoplasm; (iii) changes in ionic concentration of other small ions possibly coupled to calcium release from the SR, such as H+ or Mg++, may be monitored by other specific indicator dyes; (iv) the amount of Ca++ bound to structural regulatory sites accessible to myoplasm, for example on troponin C, may be monitored by changes in dichroism of injected dyes bound to oriented structures. By using a laser beam to illuminate a region of fiber about 1-2 Mum in diameter, it should be possible to compare the amplitudes of some of the above signals along a sarcomere (for example, comparing A-band vs. I-band amplitudes) and thereby gain evidence as to the likely structural origin of such signals. Experiments will primarily be carried out on singly-dissected, intact twitch fibers of the frog, with activity initiated by either the normal action potential mechanism or by voltage clamp. Later experiments will adapt these methods for the study of E-C coupling to other fiber types. The use of optical techniques gives promise of contributing substantially to a more quantitative understanding of the basic mechanisms that control contraction in striated muscle cells. Eventually these techniques should be useful for understanding how these mechanisms may be altered by drugs and disease.